Kynureninases are a group of pyridoxal-5'-phosphate dependent enzymes which catalyze the hydrolyric .beta.,.gamma.-cleavage of aryl-substituted .alpha.-amino-.gamma.-keto acids, particularly L-kynurenine or 3-hydroxy-L-kynurenine to give L-alanine and anthranilic acid or 3-hydroxyanthranilic acid, respectively (see: K. Soda and K. Tanizawa (1979)Advances Enzym. 49:1-40). Kynureninase is involved in the microbial catabolism of L-tryptophan via the aromatic pathway. In plants and animals, a kynureninase is required in tryptophan catabolism and for NAD biosynthesis via guinolinic acid. Quinolinic acid is a relatively toxic metabolite which has been implicated in the etiology of neurological disorders, including epilepsy and Huntington's chorea (R. Schwarcz et al. (1988) Proc. Natl. Acad. Sci. USA 85:4079; M. F. Beal et al. (1986) Nature 321:168-171; S. Mazzari et al. (1986) Brain Research 380:309-316; H. Baran and R. Schwarcz (1990) J. Neurochem. 55:738-744). Inhibitors of kynureninase are thus important targets for treatment of such neurological disorders.
L-kynurenine (which can also be designated .alpha.,2-diamino-.gamma.-oxobenzenebutanoic acid) is the preferred substrate of bacterial kynureninase, which is exemplified by that of Pseudomonas fluorescens (O. Hayaishi and R. Y. Stanier (1952) J. Biol. Chem. 195:735-740). The kynureninase of tryptophan metabolism in plants and animals has a somewhat different substrate specificity with 3-hydroxy-L-kynurenine (which can be designated .alpha.,2-diamino-3-hydroxy-.gamma.-oxobenzenebutanoic acid) being the preferred substrate (Soda and Tanizawa (1979) supra).
The mechanism of kynureninases has been the subject of considerable interest due to the unique nature of this pyridoxal-5'-phosphate dependent reaction. Mechanisms based on redox reactions (J. B. Longsnecker and E. E. Snell (1955) J. Biol. Chem. 213:229-235) or transamination (C. E. Dalgleish et al. (1951) Nature168:20-22) have been proposed. More recently mechanisms involving either a nucleophilic mechanism with an "acyl-enzyme" intermediate (C. Walsh (1979) "Enzymatic Reaction Mechanisms" W. H. Freeman and Co., San Francisco, p. 821; M. Akhtar et al. (1984) "The Chemistry of Enzyme Action" New Comprehensive Biochemistry, Vol. 6 (M. I. Page, ed.) Elsevier, New York, p.821) or a general base-catalyzed mechanism (K. Tanizawa and K. Soda (1979) J. Biochem. (Tokyo) 86:1199-1209) have been proposed.
In addition to the physiological reaction, kynureninase has been shown to catalyze an aldol-type condensation of benzaldehyde with incipient L-alanine formed from L-kynurenine to give .alpha.-amino-.gamma.-hydroxy-.gamma.-phenylbutanoic acid (G. S. Bild and J. C. Morris (1984) Arch. Biochem. Biophys. 235:41-47). The stereochemistry of the product at the .gamma.-position was not determined, although the authors suggested that only a single isomer was formed.
J. L. Stevens (1985) J. Biol. Chem 260:7945-7950 reports that rat liver kynureninase displays cysteine conjugate .beta.-lyase activity. This enzyme activity is associated with cleavage of s-cysteine conjugates of certain xenobiotics to give pyruvate, ammonia and a thiol, for example, cleavage of S-2-(benzothiazolyl)-L-cysteine to give 2-mercaptobenzothiazole, pyruvate and ammonia. More recently, I. S. Blagbrough et al. (1990) Toxicol. Lett 53(1-2):257-259 (Chem. Abstract 114(9):77537k) report that cysteine conjugate .beta.-lyase (C-S-lyase) is a member of a family of transaminases and aminotransferases and that C-S lyase is a glutamine transaminase K. The reference discusses structure-activity relations displayed by C-S-lyases. C-S-lyases are distinguishable from kynureninase but exhibit overlapping activities.
Several reports concerning the relative reactivities of kynurenine analogs with bacterial kynureninase or rat liver kynureninase are summarized in Soda and Tanizawa (1979) supra. Tanizawa and Soda (1979) supra reported that a number of ring substituted L-kynurenines, namely: 3-hydroxy-, 5-hydroxy-, 5-methyl-, 4-fluoro-, and 5-fluoro-L-kynurenine were substrates of kynureninase of P. fluorescens. These authors also reported that dihydrokynurenine (called .gamma.-(o-aminophenyl)-L-homoserine therein) was a substrate for that kynureninase, yielding o-aminobenzaldehyde and L-alanine. The K.sub.m of dihydrokynurenine was reported to be 67 .mu.M compared to a K.sub.m of 35 .mu.M for L-kynurenine and 200 .mu.M for 3-hydroxy-L-kynurenine. N'-formyl-L-kynurenine and .beta.-benzoyl-L-alanine were likewise reported to be substrates (with K.sub.m =2.2 mM and 0.16 mM, respectively) for the bacterial kynureninase. Tanizawa and Soda measured relative reactivity as relative amounts of L-alanine formed.
O. Hayaishi ( 1955 ) in "A Symposium on Amino Acid Metabolism" (W. D. McElroy and H. B. Glass, eds.) Johns Hopkins Press, Baltimore pp. 914-929 reported that 3-hydroxy- and 5-hydroxy-L-kynurenine, .beta.-benzoyl-L-alanine and .beta.-(o-hydroxybenzoyl)-L-alanine PG,5 were substrates for the bacterial enzyme, but that N'-formyl-L-kynurenine was not a substrate. O. Hayaishi measured relative reactivities by determining the amount of substrate hydrolyzed.
Tanizawa and Soda (1979) supra reported that S-benzoyl-L-cystsine, L-asparagine and D-kynurenine were not substrates of kynureninase, while O. Hayaishi (1955) supra reported that .beta.-(p-aminobenzoyl)-L-alanine, .beta.-(o-nitrobenzoyl)-L-alanine, .beta.-(m-hydroxybenzoyl)-L-alanine, 3-methoxy-L-kynurenine, .beta.-benzoylpropanoic acid, and .beta.-(o-aminobenzoyl)propanoic acid do not react with bacterial kynureninase. Kynureninase is reported to act only on L-amino acids (M. Moriguchi et al. (1973) Biochemistry 12:2969-2974).
O. Wiss and H. Fuchs (1950) Experientia 6:472 (see: Soda and Tanizawa (1979) supra) reported that 3-hydroxy-L-kynurenine, L-kynurenine, .beta.-benzoyl-L-alanine, .gamma.-phenyl-L-homoserine, .gamma.-methyl-L-homoserine, 2-aminolevulinic acid and .alpha.-amino-.gamma.-hydroxypentanoic acid reacted with rat liver kynureninase to produce alanine, while .beta.-(o-nitrobenzoyl)-L-alanins did not.
G. M. Kishore (1984) J. Biol. Chem. 259:10669-10674 has reported that certain .beta.-substituted amino acids are mechanism-based inactivators of bacterial kynureninase. Several .beta.-substituted amino acids including: .beta.-chloro-L-alanine, o-acetyl-L-serine, L-serine O-sulfate, S-(2-nitrophenyl)-L-cystsine (called S-(o-nitrophenyl)-L-cysteine, therein) and .beta.-cyano-L-alanine inactivated kynureninase. These .beta.-substituted amino acids react with kynureninase to give pyruvate and ammonia. However, a portion of the turnovers of the enzyme lead to formation of an inactive enzyme complex. S-(2-nitrophenyl)-L-cysteine was described as the "most efficient suicide substrate at low concentrations" with a K.sub.i of 0.1 mM.
Bacterial kynureninase is also strongly inhibited by o-aminobenzaldehyde (K.sub.i =6.5 .mu.M, non-competitive inhibition). Several other aromatics having "a carboxyl group on the benzene ring and an amino group at the ortho-position" including o-aminoacetophenone, anthranilic acid o-nitrobenzaldehyde and benzaldehyde were described as inhibitors (Tanizawa and Soda (1979) supra). It was suggested that inhibition relates to binding of the formyl group to the portion of the enzyme that serves as a binding site for the .gamma.-carboxyl of kynurenine. Anthranilate and 3-hydroxyanthranilate, the products of the kynureninase reaction, were also reported to inhibit the enzyme (Takeuchi et al. (1980) J. Biochem. (Tokyo) 88:987-994).
Blagbrough, I. S. et al. (1988) Drug Metab. Drug Interact 6(3-4):303-316 in them. Abstracts 112(19):174617c report on inhibition of rat renal C-S lyase by certain cystsine conjugates. Certain S-(nitro-substituted phenyl)-L-cysteines and N-acetyl-S-(nitro-substituted phenyl)-L-cysteines were reported to inhibit C-S lyase as measured by a kidney slice methodology. The nitrophenyl cystsine conjugates: S-(2-nitrophenyl)-L-cysteine, S-(4-nitrophenyl)-L-cysteine, S-(2,6-dinitrophenyl)-L-cysteine, N-acetyl-S-(3,4-dinitrophenyl)-L-cystsine, N-acetyl-S-(2,6-dinitrophenyl)-L-cysteine and N-acetyl-S-(2-chloro-4-nitrophenyl)-L-cysteine are reported to inhibit C-S layse.
Vamvakas et al. (1988) Chem. Biol. Interact 65:59-71 in Chem. Abstracts 109(7):50020w refers to the cystsine conjugate .beta.-lyase-mediated metabolism of certain cystsine conjugates including S-benzyl-L-cysteine which was reported to be cleaved to give pyruvate. The reference notes that aminooxyacetic acid is an inhibitor of the .beta.-lyase.
Tolosa et al. (1968) Mol. Biol. 2(5):769-777 [in Russian] in Chem. Abstracts 70(1):482d reported that cystsine lyase was significantly inhibited by H.sub.2 NOH and its O-substituted derivatives and that aminooxyacetic acid was the most inhibitory derivative tested.
J. P. Whitten et al. (1989) Tetrahedron Letts. 30:3649-652 reported the synthesis of 2,2-difluoro-.alpha.-benzoyl alanine (.alpha.-amino-.beta.,.beta.-difluoro-.gamma.-oxobenzene butanoic acid) which is said to be a "potential new inhibitor of kynureninase." Fluoroketone-containing peptides are described as capable of forming stable hydrates or hemiketals which are "thought to inhibit" proteolytic enzymes as analogs of a tetrahedral transition state. The difluoro compound is described as a competitive inhibitor of kynureninase, but no details of this inhibition are given in the reference.
The present work is based on a reexamination of the mechanism of kynureninase catalysis, in particular, through an investigation of the stereospecificity of the retro-aldol reaction catalyzed by the enzyme. During the course of this work, the reactivity of dihydrokynurenine with kynureninase was found to be significantly different than had previously been reported. The result of these mechanism and reactivity studies was the identification of a class of potent kynureninase inhibitors. The present invention provides kynureninase inhibitors which are designed to be "transition-state analogue" inhibitors.